Nuclear receptors, ligands and coactivators: A study of their interactions using Fluorescence Fluctuation Spectroscopy

Who:  Yan Chen - (University of Minnesota)
Where:  1080 PRB, Robert Smith Seminar Room
When:  Wednesday, November 18, 2009 at 01:00
Type:  Biophysics Seminar

Description: Nuclear receptors are ligand inducible transcription factors. Upon agonist binding, nuclear receptors recruit coactivators, and subsequently activate gene transcription. Experimental determination of the binding curve and stoichiometry of nuclear receptor and coactivator complexes in living cells is a prerequisite for quantitative modeling of this cellular process. Dual-color fluorescence fluctuation spectroscopy provides a general framework for detecting protein interactions. However, quantitative characterization of protein hetero-interactions remains a difficult task. To address this challenge we introduce hetero-species partition (HSP) analysis for measuring protein hetero-interactions of the type D + nA ↔ DA_n. HSP directly identifies the hetero-interacting species from the sample mixture and determines the binding curve and stoichiometry in the cellular environment. The method is applied to measure the ligand-dependent binding curve of the nuclear receptor retinoic X receptor to the coactivator transcription intermediate factor 2. The binding stoichiometry of nuclear receptor / coactivator complexes has never been directly measured. A previous study using protein fragments observed a higher binding stoichiometry than biologically expected. We address this difference in stoichiometry by measuring the binding curves of the full-length proteins in living cells. We also extended this technique to another nuclear receptor, peroxisome proliferator-activated receptor, and study its interactions with coactivators. Our studies provide proof-of-principle experiments that illustrate the potential of HSP as a general and robust analysis tool for the quantitative characterization of protein hetero-interactions in living cells.